Thermo Scientific Dionex CarboPac PA100 Column

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1 CHROMTOGRPHY Thermo Scientific Dionex CarboPac P Column Product Specifications HPLC Columns for Oligosaccharide Mapping and Purification Predictable, high resolution separations of oligosaccharides released from glycoproteins Neutral and sialylated N-linked oligosaccharides from glycoproteins Oligosaccharides with monosaccharide linkage isomerism Oligosaccharides in food products Linear polysaccharide profiling Thermo Scientific Dionex ERD Electrolytically Regenerated Desalter for fraction collection Predictable, High Resolution Separations of Oligosaccharides Released from Glycoproteins Characterization of oligosaccharides released from glycoproteins is an important but non-trivial task for the biotechnology and pharmaceutical industries. The Thermo Scientific Dionex CarboPac P column provides the resolution needed for routine analysis of these oligosaccharides. Neutral and charged oligosaccharides are separated in their anionic forms by highperformance anion-exchange chromatography (HPE) using the Dionex CarboPac P column. Detection of these compounds at low picomole levels has been optimized using pulsed ampero metric detection (PD). No sample derivatization is required for detection. Separations of oligosaccharides are based on their fine structural differences, such as the composition and the sequence of the oligosaccharides, linkage isomerism, degree of sialylation, and degree of branching. There are several factors affecting the elution of oligosaccharides using the CarboPac P column. Twelve empirical relationships between oligosaccharide structure and chromatographic retention are documented by Rohrer (Rohrer, J. Glycobiology, 99,, 9-6). Some of these are illustrated in Figure and include: Fucosylated oligosaccharides are eluted ahead of their afucosylated analogs (peaks and ).

2 Retention times of high mannose oligosaccharides increase as the number of mannose residues increases (peaks,, 9). s the degree of branching increases, the retention time of the oligosaccharide increases (peaks, 9, ). Removal of the terminal galactose residues from a complex oligosaccharide reduces its retention time (peaks 7 9, ). Neutral and Sialylated N-linked Oligosaccharides from Glycoproteins The elution of acidic sugars from the Dionex CarboPac P column requires stronger eluents than those used for the elution of neutral sugars. This is usually accomplished with the addition of sodium acetate to the sodium hydroxide eluent. Sodium acetate accelerates the elution of strongly bound species and offers further selectivity control, without interfering with pulsed amperometric detection. Figures and show the separation of neutral and sialylated oligosaccharides in a single run. The neutral sugars are eluted in a group at the beginning of the profile followed by the disialylated, the trisialylated, and finally the tetrasialylated oligosaccharides. Oligosaccharides with Monosaccharide Linkage Isomerism High resolution separations can be obtained based on linkage isomerism, which is difficult to achieve using other chromatography technologies. Under alkaline conditions, the technique resolves these species not only by sialic acid content, but also according to the combination of α(,)- and α(,6)- linked sialic acids within each charge class. Oligosaccharides with the greatest proportion of α(,6)- to α(,)-linked sialic acids are the least retained (Figure ). The neutral component of the oligosaccharides also influences separation: those containing a Galβ(,)GlcNc sequence are retained longer than those with Galβ(,)GlcNc. n Dionex CarboPac P + Guard mm Sodium acetate gradient in mm Sodium hydroxide Flow Rate: ml/min Inj. Volume: µl Dionex ED detector, gold electrode Peaks:. Fucosylated Man GlcNc. Man GlcNc. sialo agalacto bi, core fuc. sialo agalacto bi. Man GlcNc 6. sialo agalacto tri 7. sialo bi, core fuc Flow Rate: Detector:. sialo bi 9. sialo tri. Bisected hybrid. sialo tetra. Man 9 GlcNc Figure. Separation of neutral oligosaccharide standards. Twelve commonly occurring N linked neutral oligosaccharides are easily resolved within minutes. n Net Negative Charge I II III IV Figure. Separation of neutral and sialylated oligosaccharide standards. This is a commonly used method to obtain an overall profile of neutral and sialylated N-linked oligosaccharides released from glycoproteins. Oligosaccharides are separated into broad classes depending on their degree of sialylation. Based on the retention time windows of the oligosaccharide peaks, the degree of sialylation of the oligosaccharides can be predicted. Dionex CarboPac P mm Sodium acetate over min in mm Sodium hydroxide Flow Rate: ml/min Peaks:. Fucosylated Man GlcNc. Man GlcNc. sialo agalacto bi, core fuc. sialo agalacto bi. sialo bi, core fuc 6. sialo bi 7. Man 9 GlcNc, 9. Disialylated bi (reduced),. Trisialylated tri (reduced),. Tetrasialylated tri (reduced) Dionex CarboPac P mm Sodium acetate over min in mm Sodium hydroxide ml/min Pulsed amperometry, NN Gal GlcNc Man α-6 NN Gal GlcNc Man α-6 α-6 β- β- α- β- β- Man GlcNc GlcNc Man GlcNc GlcNc β- β- β- β- NN Gal GlcNc Man α- NN Gal GlcNc Man α- α-6 β- β- α-6 β- β- NN Gal GlcNc α- β- β- Peak NN Gal GlcNc α- β- β- Peak Figure. Separation of trisialylated oligosaccharides. The high resolving power of the Dionex CarboPac P column is demonstrated by the resolution of these two oligosaccharides, which differ by only a single linkage alpha -6 vs. alpha -.

3 Oligosaccharides in Food Products Oligosaccharides are routinely determined in food and beverage products for a variety of purposes, including quality control, verifying food-labeling claims, establishing product authenticity, and monitoring fermentation processes. Oligosaccharide profiles obtained using HPE-PD with the Dionex CarboPac P column can be used to establish the fingerprint of food samples. Suspect samples can be analyzed and compared to the known profiles. This technique is useful in detecting adulteration and in quality control. For example, oligosaccharide profiles are used to detect adulteration of natural fruit juices (Figure ), establish the geographic origin of molasses, and analyze polysaccharides in hydrolyzed glucose syrup. The Dionex CarboPac P column easily separates mono-, di-, and trisaccharides in citrus and sunflower honey (Figure ). These profiles are used as fingerprints for quality control and labeling verification purposes. ) Pure Orange Juice B) Orange Juice dulterated with Beet Medium Invert Sugar Dionex CarboPac P Sodium hydroxide/ Sodium acetate gradient Flow Rate: ml/min Inj. Volume: µl Figure. Oligosaccharide profiles of pure and adulterated orange juice. Oligosaccharide composition profiles can be used to detect the adulteration of natural fruit juices by inexpensive sweeteners such as beet sugar. Dionex CarboPac P Gradient: ( mm) + Guard t B C : 6 mm NaOH B:. M NaOc C. mω DI Water. Flow Rate:.7 ml/min Peaks:. Glycerol. α,α-trehalose. Glucose. Fructose. Melibiose 6. Isomaltose 7. Sucrose. Unknown 9. Turanose. Palatinose. Nigerose. Maltose/-Kestose. Laminaribiose. Eriose. Panose B mv 6 7 mv Seminars in Food nalysis, Vol. No. /, 997, Corrandini, C. et. al., pplication of HPE-PD to Carbohydrate nalysis in Food Products and Fruit Juices, Rapid Science LTD. Figure. Chromatograms of di- and trisaccharides in honey from () citrus and (B) sunflower.

4 Linear Polysaccharide Profiling Separations of high mannose, hybrid, and complex oligosaccharides are best accomplished using the Dionex CarboPac P column. Separations are accelerated and improved by using sodium acetate gradients in sodium hydroxide. Figure 6 shows the gradient separation of chicory inulin, illustrating the high resolution possible with this column. Flow Rate: Detector: Range: Dionex CarboPac P : mm NaOH B: mm NaOH with M NaOc. ml/min Pulsed amperometry, n full scale Sample: Gradient: µl of.%(w/v) solution t B 7 6 Determining the levels of fermentable and nonfermentable sugars at every stage of beer production is important because fermentable sugars determine the final alcohol content, and nonfermentable sugars contribute to the flavor and body of the final product. separation of maltose oligomers up to degree of polymerization (DP) is shown in Figure 7, panel ; panels B, C, and D show sugar and polysaccharide profiles at various stages of the brewing process. ll samples were diluted :. PD Response (mv) Time (min) 6 Figure 6. Gradient separation of chicory inulin using the Dionex CarboPac P. Sodium acetate, mm Dionex CarboPac P and Guard Sodium hydroxide/ Sodium acetate gradient Flow Rate:. ml/min Inj. Volume: µl Dionex ED detector, gold electrode ) Maltose Oligomers 9 C) Wort Peaks:. Ethanol. Glucose. Maltose. Maltotriose. Maltose-Oligomers 6 B) First Mash D) Finish Figure 7. Sugar and oligosaccharide profiles during beer production. In panel, maltose oligomers are baseline-separated. Chromatograms B, C, and D illustrate sugar and oligosaccharide profiles at different stages of the brewing process.

5 Fraction Collection The Dionex ERD Desalter device is a membrane device designed to desalt and reduce the ph of the samples following HPE-PD when the user wishes to collect and further analyze the carbohydrate samples. Desalted samples are then ready for lyophilization without dialysis. More than 99% of the sodium ions in eluents that contain up to. M sodium ions, flowing at a rate of ml/min, are removed by the Dionex ERD Desalter device. n advantage of the Dionex CarboPac P column over the Dionex CarboPac P column is that a lower salt concentration is required to elute the same oligosaccharide, making the Dionex CarboPac P column the best choice if fraction collection with subsequent desalting is required. Placed after the electrochemical detector, the Dionex ERD Desalter device exchanges sodium ions for hydronium ions. This process changes the sodium hydroxide and sodium acetate eluents to water and dilute acetic acid immediately after leaving the detector cell. Collected fractions can then be lyophilized, leaving the pure carbohydrate sample ready for further manipulation. These samples are suitable for enzymatic and chemical digestion, NMR or mass spectrometric analysis, or further chromatographic analysis. Figure shows the effect of the added volume of the Dionex ERD Desalter device between detection and collection points. Loss of resolution is measurable (~6%), but chromatographic resolution is sufficient to allow acceptable purification. Rugged, Reliable Separation with Guaranteed Performance The unique pellicular resin of the Dionex CarboPac P columns offers exceptional selectivity and stability over the entire ph range. Its highly crosslinked structure ensures long column life and easy clean-up. The entire manufacturing process (resin synthesis, amination, and packing and testing of the chromatographic columns) is carefully controlled to ensure that every Dionex CarboPac P column delivers reproducible performance. Dionex CarboPac P columns are tested with two isomers of N-acetylneuraminosyl-D-lactose to ensure lot-to-lot reproducibility. B 6 7 Figure. Elution of nmol fetuin N-linked alditols. Dionex CarboPac P Column 6 6 SPECIFICTIONS Detection: Sample: Dionex CarboPac P to mm Sodium acetate from to min in mm sodium hydroxide Pulsed amperometry, Dionex ED detector, gold electrode Fetuin N-linked alditols, nmol ) Detection before Dionex ERD desalter B) Detection before fraction collector Peaks:. Tri-S (la + ). Tri-S (la + lb). Tri-S (,6). Tri-S (,). Tri-S (,6) 6. Tri-S (,) Resin Composition. µm diameter ethylvinylbenzene/ divinylbenzene substrate (% crosslinking) agglomerated with 7 nm MicroBead 6% crosslinked quaternary amine-functionalized latex. nion-exchange Capacity pproximately 9 µeq/column ( mm) Maximum Operating Pressure psi ( MPa) Chemical Compatibility ph, % compatible with common organic solvents Temperature Range 9 ºC Test Procedure Separation of a sialylated N-linked fetuin alditol test mixture (P/N 6) Recommended Operating Temperature mbient Recommended Flow Rate. ml/min Ionic Form Eluents Sodium acetate and sodium hydroxide only

6 Ordering Information In the U.S., call () 6-69 or contact the Thermo Fisher Scientific Regional Office nearest you. Outside the U.S., order through your local Thermo Fisher Scientific office or distributor. Refer to the following part numbers. Thermo Fisher Scientific can also make special order Dionex CarboPac columns to your specifications; call for more information. Description Dionex CarboPac P Columns Part Number CarboPac P nalytical Column ( mm) CarboPac P Guard Column ( mm) CarboPac P Microbore Column ( mm) 7 CarboPac P Microbore Guard Column ( mm) 7 CarboPac P Semipreparative Column (9 mm) CarboPac P Preparative Column ( mm) SP9 SP667 Product Specifications Carbohydrate Membrane Desalter Dionex ERD Electrolytically Regenerated Desalter ( mm) 7 Dionex ERD Electrolytically Regenerated Desalter ( mm) 9 Thermo Fisher Scientific Inc. ll rights reserved. ISO is a trademark of the International Standards Organization. ll other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. Thermo Fisher Scientific, Sunnyvale, C US is ISO 9 Certified. frica + ustralia ustria + 6 Belgium + 7 Brazil + 7 Canada + 7 China (free call domestic) 6 PS7-EN S Denmark Europe-Other + Finland France Germany +9 6 India Italy Japan Korea + 6 Latin merica Middle East + Netherlands New Zealand Norway Russia/CIS + Singapore Sweden Switzerland Taiwan UK/Ireland + US + 7